Molecular tools-advances, opportunities and prospects for the control of parasites of veterinary importance.

The recent advancement in genome sequencing facilities, proteomics, transcriptomics, and metabolomics of eukaryotes have opened door for employment of molecular diagnostic techniques for early detection of parasites and determining target molecules for formulating control strategies. It further leads to the introduction of several purified vaccines in the field of veterinary parasitology. Earlier, the conventional diagnostic methods was entirely based upon morphological taxonomy for diagnosis of parasites but nowadays improved molecular techniques help in phylogenetic study and open an another area of molecular taxonomy of parasites with high precision. Control measures based upon targeting endosymbionts in parasites like Dirofilaria immitis is also under exploration in veterinary parasitology. Metagenomics have added an inside story of parasites bionomics which have created havoc in human and animals population since centuries. Omics era is playing a key role in opening the new approaches on parasite biology. Various newer generations of safer vaccines like edible vaccines and subunit vaccines and diagnostic techniques based upon purified immunologically active epitopes have become commercially available against the parasites (helminths, protozoa and arthropod borne diseases). Nowadays, a transgenic and gene knock out studies using RNA interference and CRISPR are also helping in understanding the functions of genes and screening of target genes, which are not available before the advent of molecular tools. Molecular techniques had paramount impact on increasing the sensitivity of diagnostic tools, epidemiological studies and more importantly in controlling these diseases. This review is about the advancements in veterinary parasitology and their impact on the control of these pathogens.

Morphological and molecular characterization of Acrobeloides saeedi Siddiqi, De Ley and Khan, 1992 (Rhabditida, Cephalobidae) from India and comments on its status.

Two cultured populations of Acrobeloides saeedi are described from India. Morphologically and morphometrically this material agrees with other species of the Maximus-group (A. bodenheimeri, A. longiuterus, and A. maximus), especially with A. longiuterus. However, molecular studies based on 18 S, 28 S and ITS rDNA confirmed the Indian material is well differentiated from all of these species. According to this, A. saeedi is considered a valid taxon distinguished mainly from A. bodenheimeri by having dextral female reproductive system (vs sinistral), from A. longiuterus by having larger females (1.03-1.57 vs 0.57-0.88 mm) and from A. maximus by having seta-like labial processes (vs absent) and males as frequent as females (vs males very infrequent). Molecular and phylogenetic studies revealed the present specimens to be conspecific to undescribed Acrobeloides sp. population from Iran, and hence, both regarded to be conspecific to each other. In addition, other similar species are revised: Acrobeloides ishraqi is considered new junior synonym of A. saeedi, Acrobeloides mushtaqi is considered new junior synonym of A. bodenheimeri, while Acrobeloides gossypia is also considered junior synonym of A. saeedi.Two cultured populations of Acrobeloides saeedi are described from India. Morphologically and morphometrically this material agrees with other species of the Maximus-group (A. bodenheimeri, A. longiuterus, and A. maximus), especially with A. longiuterus. However, molecular studies based on 18 S, 28 S and ITS rDNA confirmed the Indian material is well differentiated from all of these species. According to this, A. saeedi is considered a valid taxon distinguished mainly from A. bodenheimeri by having dextral female reproductive system (vs sinistral), from A. longiuterus by having larger females (1.03-1.57 vs 0.57-0.88 mm) and from A. maximus by having seta-like labial processes (vs absent) and males as frequent as females (vs males very infrequent). Molecular and phylogenetic studies revealed the present specimens to be conspecific to undescribed Acrobeloides sp. population from Iran, and hence, both regarded to be conspecific to each other. In addition, other similar species are revised: Acrobeloides ishraqi is considered new junior synonym of A. saeedi, Acrobeloides mushtaqi is considered new junior synonym of A. bodenheimeri, while Acrobeloides gossypia is also considered junior synonym of A. saeedi.

Morphological, morphometrical, and molecular characterization of Metarhabditis amsactae (Ali, Pervez, Andrabi, Sharma and Verma, 2011) Sudhaus, 2011 (Rhabditida, Rhabditidae) from India and proposal of Metarhabditis longicaudata as a junior synonym of M. amsactae.

A new population of Metarhabditis amsactae from India is morphologically, morphometrically, and molecularly characterized. This material is characterized by having 0.65 to 1.14 mm length, lips rounded, and grouped in pairs, stoma with metastegostoma bearing setose denticles, pharynx with metacorpus slightly swollen and fusiform, nerve ring, and excretory pore located at isthmus level, female reproductive system didelphic-amphidelphic with vulva equatorial, female tail conical-elongate with acute tip, male tail conical with large and robust posterior filiform part, spicules free with hooked manubrium slightly bent ventrad, gubernaculum with narrow corpus, bursa open leptoderan with eight genital papillae and phasmids posterior to the GP8. Molecular studies based on 18S and 28S rDNA genes are provided for the first time for the species. In addition, integrated morphological, morphometrical, and molecular characters are compared with other previous records of the species. According to our analysis, Metarhabditis longicaudata and other material described as different species are proposed as new junior synonyms of M. amsactae.

First Report and Comparative Study of Steinernema surkhetense (Rhabditida: Steinernematidae) and its Symbiont Bacteria from Subcontinental India.

Two populations (CS19 and CS20) of entomopathogenic nematodes were isolated from the soils of vegetable fields from Bijnor district, India. Based on morphological, morphometrical, and molecular studies, the nematodes were identified as Steinernema surkhetense. This work represents the first report of this species in India. The infective juveniles (IJs) showed morphometrical and morphological differences, with the original description based on longer IJs size. The IJs of the Indian isolates possess six ridges in their lateral field instead of eight reported in the original description. The analysis of ITS-rDNA sequences revealed nucleotide differences at 345, 608, and 920 positions in aligned data. No difference was observed in D2-D3 domain. The S. surkhetense COI gene was studied for the first time as well as the molecular characterization of their Xenorhabdus symbiont using the sequences of recA and gyrB genes revealing Xenorhabdus stockiae as its symbiont. These data, together with the finding of X. stockiae, suggest that this bacterium is widespread among South Asian nematodes from the "carpocapsae" group. Virulence of both isolates was tested on Spodoptera litura. The strain CS19 was capable to kill the larvae with 31.78 IJs at 72 hr, whereas CS20 needed 67.7 IJs.

Redescription and molecular characterization of Panagrellus ceylonensis (Nematoda, Rhabditida, Panagrolaimidae) from India.

A species of genus Panagrellus was discovered from a wet season form of an oriental common evening brown butterfly Melanitis leda. In this study, a detailed description of Panagrellus ceylonensis is provided including the morphometry, light microscopy and molecular (18S and 28S rDNA genes) studies. Morphological studies on the species agree with original description and characterized by having 12501481m long body in females and 9491305m in males, lateral fields with four longitudinal incisures, lip region continuous and 811 m wide, six offset lips with protruding labial sensilla, neck 124173 m long, excretory pore at the level of basal bulb, vulva post-equatorial (V = 6871), vagina anteriorly orientated with heavily muscled vaginal walls, post-vulval uterine sac 111135 m long or 1.72.6 times as long as the corresponding body diameter, tail conical elongate with an acute terminus in both sexes, spicule 7191 m long, ventrally curved having hooked manubrium and bifurcated lamina tip, lamina ventrally curved with dorsal deflexion at about 60% of spicule length, gubernaculum 2631 m long and well developed. Morphologically, the Indian population of P. ceylonensis does not show a significant difference from the type material of P. ceylonensis in the original description. For molecular studies of this species, the sequence of 18S rDNA is obtained for the first time. Phylogenetic trees based on 18S and 28S rDNA sequences are provided in this study. Additionally, bionomics and global distribution of the species of Panagrellus genus are also discussed. In conclusion, our study provides a comprehensive morphological characterisation and molecular marker sequences of 18S, and 28S genes that can be used to support future taxonomical research on this species and emphasizes the importance of combining molecular data with morphological data to describe the species accurately.

Morphological, Morphometrical and Molecular Characterization of Oscheius siddiqii Tabassum and Shahina, 2010 (Rhabditida, Rhabditidae) from India with Its Taxonomic Consequences for the Subgenus Oscheius Andrássy, 1976.

An insect parasitic nematode belonging to the genus Oscheius was recovered from the agricultural soils from the Hapur district in western Uttar Pradesh, India. Morphological studies on this species exhibited its high resemblance with two Pakistani species: Oscheius siddiqii and O. niazii. No molecular data are available for these taxa but, morphologically, both species do not differ significantly from our strains and each other. Hence, these nematodes can be considered conspecific, and the correct name for this taxon is O. siddiqii, the first described species. The phylogenetic analyses of the ITS-, 18S-, and the 28S rDNA sequences showed that O. siddiqii is a sister taxon to the group formed by Oscheius microvilli, O. myriophilus, O. safricanus, and several unidentified Oscheius species. Additionally, our analyses show that based on molecular and morphological data, the species Oscheius rugaoensis and O. microvilli cannot be distinguished from O. chongmingensis and O. myriophilus, respectively, and are thus considered junior synonyms of these taxa. Furthermore, the available data are not sufficient to evaluate the status of Oscheius basothovii and O. safricanus, which are, in consequence, considered species inquirendae. These findings highlight the necessity of the proper morphological and molecular characterisation of the described Oscheius species.

Molecular and Phenotypic Characterization of Heterorhabditis indica (Nematoda: Rhabditida) Nematodes Isolated During a Survey of Agricultural Soils in Western Uttar Pradesh, India.

INTRODUCTION: Entomopathogenic nematodes (EPNs) are important biocontrol agents of insect pests. To increase the availability of locally adapted entomopathogenic nematode isolates for biocontrol programs, a survey of several agricultural soils in Western Uttar Pradesh, India was conducted. MATERIALS AND METHODS: Eight hundred and sixty soil samples from the districts Meerut, Bulandshahr, Baghpat, and Bijnor were collected and examined for the presence of entomopathogenic nematodos using the "Galleria baiting method". Steinernema and Heterorhabditis nematodes were recovered. The isolated Heterorhabditis nematodes were molecularly, and morphologically characterized, and their biocontrol potential was evaluated against Spodoptera litura. Finally, the geographical distribution of entomopathogenic nematodes was studied based on the analysis of ITS GenBank records. RESULTS: A small proportion of the collected soil samples were positive for Heterorhabditis and Steinernema nematodes. Twelve soil samples were positive for the presence of Heterorhabditis nematodes, and 29 samples were positive for Steinernema. The Heterorhabditis nematodes were identified as Heterorhabditis indica based on morphological, morphometrical and molecular analyses. No other species of Heterorhabditis were isolated from the soil samples analyzed, suggesting that this species is dominant in the western part of Uttar Pradesh, India. The morphology of the nematode isolates was somewhat similar to the morphology of the H. indica isolate used for the original description of this species, with a notable exception mucrons were present in the hermaphrodite and female specimens we collected, but this structure was not observed in the specimens used for the original description of the species. Principal component analyses (PCA) show small inter- and intraspecific morphological variability between the nematodes species of the "Indica" clade. The insecticide properties of one isolate, CH7, were evaluated against Spodoptera litura, and the results show that this isolate effectively killed this pest under laboratory conditions, demonstrating its potential as a biocontrol agent. CONCLUSION: This study sets the basis for establishing new biocontrol agents to be used in future pest management programs in India.

Morphological and molecular characterization of Heterorhabditis bacteriophora isolated from Indian soils and their biocontrol potential.

Agricultural soils and open fields from Western Uttar Pradesh (India) were surveyed to determine the presence of entomopathogenic nematodes. From the entomopathogenic nematodes isolated, Heterorhabditis isolates were selected and further characterized using morphological, morphometrical and molecular approaches. The results showed that three isolated nematodes were Heterorhabditis bacteriophora and were associated with Photorhabdus laumondii subsp. clarkei bacteria, while the rests were identified as Heterorhabditis indica. The biocontrol potential of H. bacteriophora against three agricultural pests was evaluated. Nematode infectivity experiments showed that the nematode isolates DH7 and DH8 were highly pathogenic against cotton bollworm (Helicoverpa armigera) and tobacco cutworm (Spodoptera litura), and less pathogenic against white grub (Holotrichia serrata) larvae. This study sets the basis for establishing new biocontrol agents to be used in pest management programs in India.

Study of Steinernema hermaphroditum (Nematoda, Rhabditida), from the West Uttar Pradesh, India.

INTRODUCTION: The entomopathogenic nematodes have been reported from all continents (except Antarctica) and almost all regions of the world. Surveys of EPNs in India has resulted in the recovery of several isolates of Steinernema. Among one of them, isolate CS34 was identified as S. hermaphroditum Stock, Griffin & Chaerani, 2004. We investigated the identification and the pathogenicity of S. hermaphroditum in District Meerut of Western Uttar Pradesh, India. MATERIALS AND METHODS: The Steinernema was examined for its pathogenicity and accurate identification by the mean of morphological and molecular technique and its geographical distribution was mapped based on meta-analysis of the ITS GenBank records. RESULTS: The surveys of agricultural soils of district Meerut, India, resulted in the isolation of one strain from entomopathogenic nematode labelled CS34 through Galleria baiting technique. Morphological characters and morphometrical analysis indicated that the strain CS34 was closely related to the "glaseri" group of Steinernema spp. The Nblast results indicated that ITS rDNA sequence had no nucleotide differences in comparison with the S. hermaphroditum (JQ687355). However, one variation in the D2-D3 segment of 28S rDNA was observed in comparison with the AY598358. The phylogenetic analysis using ITS and 28S rDNA indicated that the Indian S. hermaphroditum could be placed together with other S. hermaphroditum, with strong posterior probability. Besides, the PCA analysis demonstrated some variability within the test populations. The distribution of S. hermaphroditum based on meta-analysis of the GenBank records showed its presence in the three Asian countries-India, Thailand and Indonesia. The Indian strain of S. hermaphroditum also tested positively for its virulence against three major pests, namely, Galleria mellonella, Helicoverpa armigera, and Spodoptera litura, with resultant which showed good efficacy on the mortalities. CONCLUSIONS: In conclusion, the economy of India is agriculture-based, but there are huge losses due to different insect pests infesting different crops. Steinernema hermaphroditum CS34 is an indigenous species to Indian subcontinent and efforts should be made to evaluate its virulence and pathogenicity against the other agricultural pests hampering productivity throughout the country. This may lead to incorporate S. hermaphroditum strain CS34 as a regular biological control agent against important lepidopteran pest in integrated pest management programs in the future.